Neurofilament proteins (NFPs) are found exclusively in the nervous system and contain (especially in middle, NF-M and high, NF-H subunits) large numbers of serine phosphorylation sites. Multiple kinase systems are operative in phosphorylating these sites. Our initial aim is to identify these specific kinases in the nervous system. In this regard, we have characterized the following three different classes of second messenger- independent kinases from rat spinal cord which phosphorylate NFPs: 1) casein kinase I (CKI)-like activity appears to be associated with NF preparations isolated from rat spinal cord. CKI, purified from rat spinal cord, phosphorylates all three NF-subunits with a preference for NF-H. Casein kinase II (CKII), on the other hand, poorly phosphorylates NF-H but has higher affinity for NF-M and NF-L; 2) Microtubule-associated protein kinase (MAP kinase)-like activity, phosphorylates all three subunits. However, these kinases do not phosphorylate dephosphorylated NFs, nor a tandemly repeated sequence motif containing lys-ser-pro (KSP) present in NF-H and NF-M where these serine residues are extensively phosphorylated; 3) P34cdc2-like kinase (KSP) kinase): this kinase appears to phosphorylate some of the multiple repeats of KSP sequences present in NF-M and NF-H. The consensus sequence recognized by this kinase is XS/TPXK. The properties of this kinase show that it is closely related to P34cdc2 kinase which is known to be involved in the regulation of the cell cycle. This observation implies a different role for neuronal cdc2- like kinase in the nervous system where cell division is minimal. We have also taken a molecular biological approach to study the cdc2-like kinase in the nervous system and have cloned and structurally characterized a P34cdc2-like kinase. A low stringency screening procedure was used to screen a rat brain cDNA library using a mouse P34cdc2 cDNA as a probe. A cDNA was identified and named neuronal cdc2- like kinase (nclk) based on its sequence similarity to P34cdc2 and its predominantly neuronal expression.